* Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes

+

* Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.

+

* Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.

+

* Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 &mu;l tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 &mu;l.

+

* Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.

+

* Redissolve the DNA pellet in 100 &mu;l TE (this will take an hour or so).

+

* Spin down the tube briefly and measure OD and 260/280 ratios.

+

* Expect around 500 ng/&mu;l concentration (total 50 &mu;g).

-

Add 15 &mu;l of SDS 20% solution

-

Incubate at 37&deg; for 1 hour

+

A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.

Add an equal volume (1.2 ml) of chloroform/isoamyl alcohol 24:1, vortex and spin down at 7000g for 6 minutes

+

==Qiagen Genomic Tip protocol==

-

Remove the supernatent to a fresh tube and add an equal volume of phenol/chloroform/isoamyl alcohol 25:24:1 and mix carefully.

+

For buffers, see [[Qiagen Buffers]].

-

Spin down at 7000g for 6 minutes

+

* Grow 40 ml cultures of the Mesoplasma species at 30&deg;C in 1161 medium.

+

* Pellet cells at 5000 x g for 10 minutes

+

* Resuspend the pellet in 11 ml of buffer B1 (with RNAse)

+

* Add 500 &mu;l of proteinase-K solution, 20 mg/ml (10 mg)

+

* incubate at 37&deg;C for at least 30 minutes

+

* Add 4 ml of buffer B2 and mix or vortex briefly

+

* Incubate at 50&deg;C for 30 minutes; the lysate should become clear

+

* If necessary, pellet particulates

+

* Save a 300 &mu;l sample 1

-

Remove the supernatent to a fresh tube and add an equal volume of chloroform/isoamyl alcohol 24:1 and mix carefully.

+

* Equilibrate a genomic tip 500/G with 10 ml of buffer QBT and allow flow through

+

* Vortex the sample for 10-20 seconds to reduce viscosity

+

* Sample should flow through, or can be diluted with buffer QBT before loading

+

* limit flow to 20-40 drops/min under pressure

+

* save a 1200 &mu;l sample of the flow through -- sample 2

-

Spin down at 7000g for 6 minutes

+

* Wash the column with 15 ml of buffer QC twice or more

+

* save a 600 &mu;l sample of the flow through -- sample 3

-

Remove the supernatent to a fresh tube and add 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution. Hook the DNA with a sterile pasteur pipet, drain, and tranfer to a fresh tube, or alternatively, spin down at high speed and pour off the supernatent.

+

* Elute with 15 ml of buffer QF prewarmed to 50&deg;C

+

* save a 600 &mu;l sample of the elution -- sample 4

-

Fill the tube with 70% ethanol to wash, and spin down at 7000g for 6 minutes.

+

* precipitate DNA by adding 10.5 ml (0.7 volumes) of isopropanol

+

* invert the tube several times and recover by spooling on a glass rod or

Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes

Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.

Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.

Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl.

Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.

Redissolve the DNA pellet in 100 μl TE (this will take an hour or so).

Spin down the tube briefly and measure OD and 260/280 ratios.

Expect around 500 ng/μl concentration (total 50 μg).

A critical element is the NaCl concentration prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in solution.